Study On TelG4 Activity Of Bos Taurus DHX36 Helicase And Its Mutants

Helicase is a type of molecular motor protein that can catalyze the chemical energy released by the hydrolysis of nucleoside triphosphate（ATP）to complete the process of moving on DNA or RNA strands and unwinding the double strands.Many life activities in the organism require the participation of helicase.DHX36 is a member of the DEAH/RHA subfamily in the superfamily 2.It is an RNA helicase with high affinity for the G4 substrate.It has received extensive attention in recent years and has achieved many structural results.These results are the unwinding mechanism and enzymatic characteristics of DHX36 helicase laid important significance.The G4 structure is a four-stranded helical structure formed by continuous Guanine DNA sequences.The formation and stability of G4 are usually dependent on metal ions.Such sequences exist widely in organism;participate in DNA replication,transcription,translation and other activities.The improper formation of G4 structure will bring a series of diseases.Some helicases with G4 structure as specific substrates are important participants in helicating G4 chain structure.Therefore,it is very important to study the interaction between these helicases and G4.In this study,Bos tauras DHX36（Bos tauras DHX36）helicase was used as a material,and the protein prokaryotic expression system was used to obtain high-purity BtDHX36 protein and its mutant protein.Fluorescence anisotropy and Stop flow were used to analyze the difference in affinity and unwinding activity o f BtDHX36 protein and its mutant protein to TelG4.To provide a reference for the in-depth study of the enzymatic activity of the DEAH/RHA subfamily,the main achievements are as follows:（1）The extra Eco R I cleavage site of the BtDHX36 helicase gene was mutated back to the normal gene,and recombinantly constructed into the p ET15b-sumo vector to form the recombinant vector p ET15b-sumo-DHX36.After the identification was correct,it was transferred to the expression strain BL21（DE3）soluble expression.Eluted by N i-NTA affinity chromatography,digested by sumo,and eluted by SP column can obtain the target protein with purity>95%.The molecular weight is 108k Da.（2）Using fluorescence anisotropy method as reference factors for KC l,Mg C l₂concentration,p H value and reaction temperature,etc.when exploring the optimal substrate binding conditions of BtDHX36 helicase in vitro,the optimal substrate binding conditions of BtDHX36 helicase in vitro were 50 mmol/L KC l,2 mmol/L Mg C l2,20 mmol/L Tris-HC l=7.5,reaction temperature 37℃,under this condition,BtDHX36 was found to have a high affinity for G-rich sequences.After the composite crystallization of BtDHX36 and Poly（GT）,the conditions for crystallization can be selected。（3）First,amplify the gene fragment of BtDHX36,introduce point mutation through O verlap PCR,construct the recombinant expression vector of the mutant,and after the identification is correct,purify and express,the mutant protein with purity of>95%can be obtained.（4）The Fluorescence anisotropy was used to analyze the effect of site mutations on TelG4 binding activity.The results showed that the N-terminal mutants BtDHX36^R63AI65A,BtDHX36^Y69A,BtDHX36^K76AN77AK78A were not much different from the wild-type protein,and the mutation of Y862 at the OB domain reduced the protein binding ratio to TelG4.（5）The rapid-stop fluorescence resonance energy transfer technique was used to determine the untwisting activity of BtDHX36 helicase mutant protein against TelG4-16T.The results showed that the mutation of Y862 locus of OB domain had no obvious effect on unwinding,indicating that this locus may not directly participate in the untwisting activity of BtDHX36.The N-terminal sites R63,I65,and Y69 have a significant effect on unwinding.After mutation,the proportion and rate of protein unwinding TelG4 substrate are reduced.It shows that these sites are important sites involved in TelG4 unwinding.